Work over the past year in this project has largely focused on the role of non-specific mediators of the immune and inflammatory responses in the pathogenesis of HIV-1 infection. We had previously demonstrated that Interleukin (IL)-27, a member of the IL-12 cytokine family that plays an important and diverse role in the function of the immune system, is an anti-viral cytokine capable of inhibiting HIV-1, HIV-2, Influenza virus and herpes simplex virus infection. In attempting to discern the mechanism underlying this anti-viral effect we looked at the ability of IL-27 to enhance the generation of reactive oxygen species (ROS) during the differentiation of monocytes to macrophages. Real time PCR, western blot and knock down assays demonstrated that IL-27 was able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells in association with the induction and phosphorylation of p47phox, a cytosolic component of the ROS producing enzyme, NADPH oxidase. Prior work from our lab identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic nucleic acid sensor that induces the production of interferon-lambda (IFN-L) by human primary cells and cell lines. IFN-L is a type III IFN and has similar activity to that of the type I IFNs (IFN-alpha and IFN-beta). The type I interferons are known to be a significant component of HIV-1 associated immune activation. Given our other recent observation that incomplete proviruses may be capable of leading to transcription of RNA we sought to further study the relationships between nucleic acid sensing in the cytoplasm and induction of inflammatory responses. We observed that human embryonic kidney (HEK) 293T cells, that were deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-L in response to DNA. Conversely, parental HEK 293 cells produced IFN-1 after they were exposed to exogenous DNA; however, when STING was knocked out in these cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. The DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-L in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus-2). Together, these data suggest that STING mediates the Ku70-mediated IFN-1 innate immune response to exogenous DNA or DNA virus infection. Studies are underway to see if the same occurs in the presence of defective HIV-1 proviruses. IL-15 is a pleiotropic cytokine capable of inducing the activation, proliferation and differentiation of a variety of immune cells. It has been postulated to play an important role in HIV-1 infection, yet there are conflicting reports regarding its expression levels in these patients. Over the past year we measured the levels of IL-15 in a cohort of 501 well-characterized HIV-1 infected patients and correlated the levels with plasma levels of HIV-1 and other well-known markers of inflammation, including CRP, D-dimer, sCD163 and sCD14. IL-15 levels were significantly higher in patients with viral loads >100,000 copies/ml (3.02 1.53 pg/ml) compared to either uninfected controls (1.69 0.37 pg/ml, p<0.001) or patients with a viral load <50 copies/ml (1.59 0.40 pg/ml (p<0.001). There was a significant correlation between HIV-1 viremia and IL-15 levels (Spearman r = 0.54, p<0.001) and between CD4+ T cell counts and IL-15 levels (Spearman r = -0.56, p<0.001). Thus, a significant direct correlation was noted between IL-15 levels and HIV-1 viremia and an inverse correlation was notd between IL-15 levels and CD4+ T cell counts. These data support a potential role for IL-15 in the pathogenesis of HIV-associated immune activation.